Our perception of sound relies on the expression and specific localization of a precise array of ion channels in the auditory receptors called hair cells. The long-term goal of the proposed work is to elucidate the molecular determinants of the localization and functioning of ion channels relevant to inhibitory feedback of the mammalian cochlea. The inhibitory feedback from the brainstem to the base of the outer hair cells is largely mediated by efferent nerve fibers that release Acetylcholine (ACh) and subsequently activate a K+ conductance. We have recently cloned a small-conductance K+ channel (SK2) from the mouse cochlea and obtained mouse ACh receptors and expressed them functionally in heterologous systems. Additionally, our preliminary data demonstrated that spliced variants of the SK2 channel are expressed in the cochlea and may confer specific functions. We therefore hypothesize that the SK channels and the ACh alpha9/alpha10 receptors are co-localized at the base of outer hair cells and that their co-localization involves their direct association with other protein(s). We further propose that differential splicing of SK2 channels modulates proper inhibitory feedback of the mammalian cochlea. Because the spliced variant identified lacks the Calmodulin binding domain, the channel may remain continuously activated. Thus, we propose that this spliced variant may be up regulated during noise exposure to induce membrane hyperpolarization, serving as a protective mechanism against noise over-stimulation. To test these hypotheses we will: (i) Clone, functionally express and study cochlear-specific SK channels and ACh receptors. We will study the gating, permeation, and pharmacology of the channels (SK2 and the spliced variant) as well as the properties of Ca 2+- vs voltage-dependent activation and single-channel current characteristics using patch-clamp techniques; (ii) Using a yeast two hybrid system, we will identify proteins responsible for the co-localization and possible interaction of SK channels and ACh receptors; (iii) we will monitor the expression levels and cellular distribution of spliced variants of the SK channel and study their roles in the modulation of the effects of noise in the cochlea. These studies will provide insight in to the mechanisms of efferent control of the cochlea and may help direct the design of clinical therapies to alleviate noise-induced hearing loss.